Gene expression from the delayed early 39K promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) is regulated by both a trans-activating viral factor and a cis-acting enhancer. The trans-activating factor is IE1, an immediate early gene product absolutely required for 39K expression in a transient assay analysis, but insufficient for optimal expression under certain conditions. When the concentration of IE1 is limiting, a viral enhancer element cis-linked to the 39K promoter is also required. Enhancer function is also dependent upon IE1. Thus, the IE1 gene product plays two roles in the regulation of this delayed early gene; in trans-activates both the 39K promoter and the viral enhancer that cis-activates the 39k promoter. The mechanism of action of the enhancer-dependent and enhancer-independent regulation of 39K will be defined. Specifically, this work includes five parts: (1) identification of the enhancer sequences which influence the interactions between viral DNA and trans- activating host and viral factors, (2) the identification of factors which bind the viral enhancer in normal Spodoptera frugiperda cells (host specific factors) and in cells which express the viral IE1 gene (IE1-specific factors), (3) analysis of the 39K promoter sequences that are important for transcription in the presence and absence of the cis-linked enhancer, (4) identification of factors which bind the 39K promoter in normal and in IE1- expressing cells, and (5) confirmation of in vitro binding analysis by transient assay analysis and in vivo susceptibility of viral DNA to chemical and enzymatic probes.